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Home , Nitrate, Nitrite, Nitroxyl, Oxidation state, Peroxynitrite

and its anion in aqueous solutions: Spin states, protic equilibria, and reactivities toward and nitric

Vladimir Shafirovich*† and Sergei V. Lymar†‡

* Department and Radiation and Solid-State Laboratory, New York University, New York, NY 10003; and ‡Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973

Communicated by Norman Sutin, Brookhaven National Laboratory, Upton, NY, April 5, 2002 (received for review January 28, 2002) The thermodynamic properties of aqueous nitroxyl (HNO) and its However, , which is the decomposition ؊ Ϫ anion (NO ) have been revised to show that the ground state of product, was not detected among the end products of HN2O3 NO؊ is triplet and that HNO in its singlet ground state has much decay (12). This result was interpreted as evidence against the -lower acidity, pKa(1HNO͞3NO؊) Ϸ 11.4, than previously believed. occurrence of reaction 2. On the other hand, the same research 2Ϫ These conclusions are in accord with the observed large differences ers reported peroxynitrite formation during N2O3 photolysis in ؊ 3 1 between HNO and NO in their reactivities toward O2 and NO. alkaline solution (13). To reconcile the data, it was suggested Laser flash photolysis was used to generate 1HNO and 3NO؊ by that thermal reaction 1 followed by deprotonation of HNO photochemical cleavage of trioxodinitrate (Angeli’s anion). The produces singlet NOϪ, which is the ground state in , and ؊ 1 Ϫ 3 3 spin-allowed addition of O2 to NO produced peroxynitrite with that NO is unreactive toward O . In contrast, photochemical Ϫ 2 ؋ 9 ؊1⅐ ؊1 2 ؍ nearly diffusion-controlled rate (k 2.7 10 M s ). In contrast, cleavage of N2O3 was thought to generate the long-lived triplet 3 1 Ϫ the spin-forbidden addition of O2 to HNO was not detected (k ϽϽ excited state of NO , which reacted with O2. However, it seems ؋ 105 M؊1⅐s؊1). Both 1HNO and 3NO؊ reacted sequentially with unlikely that hydration can reverse a gas-phase energy gap of 3 ؊ ͞ 3 Ϫ two NO to generate N3O3 as a long-lived intermediate; the rate about 70 kJ mol between the ground state NO and the excited ؊ 1 Ϫ 1 laws of N3O3 formation were linear in concentrations of NO and state NO (10). Moreover, by analogy with O2, whose lifetime ؋ 109 in water is only 4 ␮s (14), the existence of a long-lived excited 2.3 ؍ ؋ 106 M؊1⅐s؊1)orNOand3NO؊ (k 5.8 ؍ 1HNO (k Ϫ M؊1⅐s؊1). Catalysis by the was observed for the state of NO also appears highly unlikely. Thus, reaction 2 1 remains obscure, particularly because others have reported O reactions of HNO with both O2 and NO. This effect is explicable by Ϫ 2 ,؋ 4 ؊1⅐ ؊1 consumption during spontaneous decomposition of HN O (2 ؍ ؊ a spin-forbidden deprotonation by OH (k 4.9 10 M s )of Ϫ 2 3 ؊ 3 1 the relatively unreactive HNO into the extremely reactive NO . 15) and detection of small amounts of NO3 upon completion of 1 Dimerization of HNO to produce N2O occurred much more slowly the reaction (16). ؋ 106 M؊1⅐s؊1) than previously suggested. The implications In the early 1970s two research groups used pulse radiolysis to 8 ؍ k) Ϫ of these results for evaluating the biological roles of nitroxyl are produce HNO͞NO by reaction between the hydrated discussed. and NO (17, 18). This approach precluded examination of HNO͞NOϪ reactivities toward O because of a very rapid 2 Ϫ addition of two NO radicals to form N O , which then slowly itroxyl (HNO, also known as nitrosyl ) and its anion, 3 3 Ϫ decomposed to and NNO , are the simplest with in the ϩ1 Ϫ Ϫ Ϫ and yet their aqueous chemistry is not well NO ϩ 2NO 3 N O 3 N O ϩ NO . [3] understood. Recent suggestions that these neighbors of 3 3 2 2 the biologically important NO radical may play a role in cellular A pKa value of 4.7 for HNO was reported, although the spin (1–4) and in aerobic environments may be precur- states of NOϪ and HNO were not specified (17). The estimates Ϫ sors to cytotoxic peroxynitrite, ONOO , (5, 6) have engendered for the of the NO͞NOϪ couple based on this Ϫ considerable interest in the chemistry of HNO͞NO . The value (19) are almost a volt higher than those obtained by characterization of these species is complicated by their insta- electrochemical techniques (20, 21); this discrepancy has never bility with respect to formation of nitrous oxide (7, 8). In most been addressed. Recent ab initio calculations placed the pKa of cases where nitroxyl has been invoked as an intermediate, the HNO at 7.2 (22). rate-determining step was its generation, a situation that allows In the hope of clarifying the HNO͞NOϪ chemistry, we have Ϫ little insight into the properties and reactivities of HNO͞NO used UV laser flash photolysis to produce HNO͞NOϪ species by Ϫ themselves. The NO anion is isoelectronic with O2 and, like O2, photochemical cleavage of Angeli’s anion and to investigate their should have a triplet ground state, whereas the ground state of reactivities. Here we present evidence that HNO has a much HNO should be a singlet. Indeed, these ground state assignments weaker acidity than previously believed. We show that the have been well established for HNO͞NOϪ in the gas phase deprotonation of HNO is a slow spin-forbidden process that (9, 10). controls the observed chemistry in alkaline solutions. The Ϫ Ϫ A frequently used source for aqueous HNO͞NO is trioxo- reactivities of HNO and NO toward O2 and NO have been 2Ϫ dinitrate (N2O3 , also known as Angeli’s anion), whose conju- investigated and a quantitative mechanistic description of these gate (H2N2O3) has consecutive pKa values of 2.5 and 9.7 reactions is presented. (11). It is widely accepted (7, 8) that slow decomposition of the monoprotonated anion occurs through heterolytic NON bond Materials and Methods cleavage Sample Solutions. All chemicals were of analytical grade and were used as received. Milli-Q purified water was used throughout. Ϫ 3 ϩ Ϫ HN2O3 HNO NO2 . [1] Stock solutions of Na2N2O3 (Cayman Chemical, Ann Arbor, MI) 2Ϫ in 10 mM NaOH were prepared daily. Relatively stable N2O3 Subsequent addition of O2 could yield peroxynitrite Ϫ Ϫ ͞ ϩ 3 ͞ † HNO NO O2 ONOOH ONOO . [2] To whom reprint requests should be addressed. E-mail: [email protected] or [email protected].

7340–7345 ͉ PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.112202099 Downloaded by guest on September 23, 2021 0 1 ϭ ͞ ⅐ value and the tabulated entropy S ( HNOgas) 220.7 J (mol K) ⌬ 0 1 ϭ ͞ (27), we calculate fG ( HNOgas) 120 kJ mol. The free energy of HNO hydration is unknown, but is expected to be small by analogy with neutral molecules of similar dimensions and com- ⌬ 0 positions, e.g., HCN and H2CO, for which hydrG are approx- imately Ϫ5 and Ϫ1.7 kJ͞mol, respectively. The value ⌬ 0 1 Ϸ ͞ fG ( HNOaq) 115 kJ mol is, therefore, a reasonable estimate, which is also close to a 109 kJ͞mol value derived previously (19). The reduction potential of NO measured by the photoelec- trochemical technique has been reported as E0(NO͞NOϪ) ϭ Ϫ0.81 V vs. NHE without specifying the NOϪ spin state (21). This value is consistent with the upper limit E0(NO͞NOϪ) Ͻ Ϫ0.7 V vs. NHE that can be inferred from the onset of the irreversible NO reduction wave observed in controlled-potential coulometry (20). In both experiments the reducing electrons Fig. 1. Energy diagram for NO͞HNO͞NOϪ species in aqueous solution at 298 were supplied by the electrodes, a process for which there K and 1 mol͞kg standard states. Note the energy axis break at 120 kJ͞mol. The is no spin prohibition regardless of the product spin state. spectroscopic designations for the electronic states are given in parenthesis. Assuming, therefore, that these measurements pertain to the NO Ϫ Ϫ Ϫ 3 ⌬ 0 3 Ϸ ͞ Only the lowest excited states are shown for HNO and NO . reduction in the ground NO state, fG ( NOaq) 180 kJ mol ⌬ 0 3 Ϸ can be estimated. Finally, the values for fG ( HNOaq) 190 ͞ ⌬ 0 1 Ϫ Ϸ ͞ kJ mol and for fG ( NOaq) 248 kJ mol can be assigned under ␧ ϭ Ϫ1⅐ Ϫ1 [ 248 8,300 M cm (23)] sample solutions at pH 11–14.3 Ϫ a reasonable presumption that hydration does not appreciably 3 were prepared by diluting the Na2N2O3 stock. Unstable HN2O3 alter the gas-phase energy gaps of 75 kJ͞mol between HNO and ␧ ϭ Ϫ1⅐ Ϫ1 1 1 Ϫ 3 Ϫ [ 237 6,100 M cm (24)] sample solutions at pH 4–10 were HNO (9) and 68 kJ͞mol between NO and NO (10). 1 3 Ϫ prepared by flow-mixing equal volumes of the Na2N2O3 stock From the energy diagram, we estimate pKa( HNO͞ NO ) and 0.2 M phosphate, acetate, or borate buffers as described Ϸ11.4 and pKa(1HNO͞1NOϪ) Ϸ23, i.e., the acidity of HNO is below. (Matheson) was purified by passing through very low. This result attests to the closer chemical similarity a scrubbing column with 2 M KOH and then through water. The between HNO and an than between HNO and an ͞ ͞ various NO Ar and O2 Ar mixtures were produced by combin- . In its triplet state, HNO becomes a strong acid, 3 ͞3 Ϫ ϷϪ ing the gas streams of NO or O2 with Ar passed through pKa( HNO NO ) 1.8. calibrated flowmeters. All solutions were thoroughly purged with before introducing the NO-containing mixtures. The Peroxynitrite Formation upon Addition of O2. Alkaline solutions. In ϭ NO and O2 at 1 atm (1 atm 101.3 kPa) were taken strongly alkaline solutions trioxodinitrate is completely depro- tonated and stable for hours. Steady-state UV photolysis of as 1.9 and 1.3 mM, respectively. A 100-W arc beam Ϫ o 2 reflected at 45 from a broadband dielectric mirror (220–300 N2O3 in air-equilibrated solutions resulted in the rapid disap- 2Ϫ nm) was used for UV steady-state photolysis. All experiments pearance of the N2O3 absorption band at 248 nm and in the were performed at ambient temperature (22 Ϯ 2°C). simultaneous appearance of characteristic absorption of the peroxynitrite anion (ONOOϪ) around 300 nm and nitrite at ␭ Ͻ Laser Flash Photolysis with Flow Premixing. Transient absorption 230 nm (see Fig. 6, which is published as supporting information spectra were recorded by using a computerized kinetic spec- on the PNAS web site, www.pnas.org). This result is in qualitative trometer system as described (25). Briefly, a 266-nm Nd:YAG accord with the previous communication (13). The same spectral laser operating at 20 Hz was used to photolyse the samples in a changes were observed in O2-saturated solutions. The presence 0.4 ϫ 1-cm quartz flow cell. To allow for solution replacement of two well-defined isosbestic points at 225 and 283 nm indicated between the laser shots and monitoring slower kinetics, the the constancy of reaction during the photolysis. sample excitation frequency was reduced to either 1 or 0.1 Hz by From the spectral changes, a chemical yield of peroxynitrite Ϫ⌬ Ϫ ͞⌬ 2Ϫ using a computer-controlled electromechanical shutter. Two (defined as [ONOO ] [N2O3 ]) of 0.95 was calculated, i.e., 2Ϫ within the uncertainties in molar absorptivities used for this solutions (e.g., alkaline N2O3 and buffer), each saturated with a desired gas mixture, were forced by a positive gas pressure into estimate, the stoichiometry of photochemical reaction was a 12-jet tangential mixer and then through the flow cell with a 2Ϫ ϩ ͑ϩ ͒ 3 Ϫ ϩ Ϫ Ϸ ͞ N2O3 O2 hv ONOO NO2 . [4] flow rate of 12 ml min; the laser excitation occurred within less CHEMISTRY than1saftermixing. The transient absorption was probed along 2Ϫ Purging O2 from the solution did not alter the rate of N2O3 a 1-cm optical path by a light beam from a 75-W xenon arc lamp, decay, but completely suppressed ONOOϪ formation. These which was pulsed for short time scales, and the kinetic traces observations suggest that ONOOϪ is produced by the reaction of obtained were averaged over 10–20 laser pulses. For the quan- 2Ϫ O2 with the nascent products of the N2O3 photochemical ͞ 2 2Ϫ tum yield estimates, the laser pulse energies (30–40 mJ cm ) decomposition, rather than by direct scavenging of the N2O3 were measured by a calibrated thermoelectric bolometer. excited state by O2. This conclusion is in accord with the kinetics of peroxynitrite Results and Discussion 2Ϫ formation, which was monitored by flash photolysis of N2O3 in Energetics and Spin States. 2Ϫ To facilitate the analysis of the kinetic O2-saturated solutions. The prompt bleaching of the N2O3 data, we begin with a thermodynamic description of the inter- spectrum at ␭ Ͻ 300 nm induced by the laser flash was followed mediates that can be generated by photolysis of the trioxodini- by the absorption growth around 300 nm, corresponding to trate solutions. Fig. 1 presents the free energies of formation, ONOOϪ formation. At pH Ն12, the rise of absorbance con- ⌬ 0 ͞ ͞ Ϫ ⌬ ϭ⌬ Ϫ fG , for the NO HNO NO species in aqueous solution, formed well to a single exponential growth, At Aϱ{1 Ϫ estimated by using published data. The accurate value for exp( kformt)}. The values of kform, determined from kinetic ⌬ 0 ϭ ͞ fG (NOaq) 102 kJ mol has been derived (19). The enthalpy traces recorded at different pH values, sharply increased with the 1 of HNO formation in the gas phase has been reviewed and the solution alkalinity as shown in Fig. 2. At pH 12.5 and above, kform value of 107 kJ͞mol has been recommended (26). From this was essentially proportional to [OHϪ]. At the same time, the

Shafirovich and Lymar PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7341 Downloaded by guest on September 23, 2021 steady-state photolysis, where the transitory concentration of 1HNO is much smaller than in the flash photolysis experiments. We therefore conclude that the reaction competing with reaction 7 is a bimolecular recombination of 1HNO

1 ϩ 1 3 ϩ HNO HNO N2O H2O. [9] This reaction has been invariably invoked previously to explain the stoichiometry and the isotopic composition of end products of the spontaneous decay of Angeli’s (7, 8, 28). The competition between reactions 7 and 9 is readily amenable to analytical solution, which gives dependencies of the relative amplitude and the ONOOϪ formation rate constant upon Ϫ 1 k7[OH ] and k9[ HNO]0 (see Note 1, which is published as supporting information on the PNAS web site). The initial 1 Ϸ concentration [ HNO]0 0.05 mM was determined from the Fig. 2. Dependencies on the solution pH of the peroxynitrite formation rate peroxynitrite signal amplitudes around pH 14, where it was constant (kform, E) and the normalized absorbance amplitude recorded at 315 Ϫ Ϫ 1 ϭ ϱ nm after completion of the reaction (ᮀ). Conditions were: 0.15 mM N O2 in assumed that [ HNO]0 [ONOO ] . Fitting the theoretical 2 3 ϭ Ϯ ϫ 4 O -saturated solution; pH Ն 11 were maintained with NaOH, pH Ͻ 11 with 0.1 dependencies to the data in Fig. 2 yielded k7 (4.9 0.5) 10 2 Ϫ Ϫ Ϫ Ϫ 4 1⅐ 1 ϭ Ϯ ϫ 6 1⅐ 1 M borate. The solid lines show theoretical dependencies with k7 ϭ 4.9 ϫ 10 M s and k9 (8 3) 10 M s . The value of k7 closely Ϫ1 Ϫ1 6 Ϫ1 Ϫ1 1 Ͼ M ⅐s , k9 ϭ 8 ϫ 10 M ⅐s , and [ HNO]0 ϭ 0.05 mM, calculated as described corresponds to the slope of the kform vs. pH curve at pH 13 in in the text and Note 1. Fig. 2 and is rather insensitive to the value of k9. In contrast, the position of the inflection point in the calculated relative ampli- ⌬ tude curve is sensitive to the magnitude of k9; this, together with peroxynitrite signal amplitude at the end of the kinetic run ( Aϱ) the significant scatter of the data points, results in the relatively showed a sigmoidal dependence on pH (Fig. 2). A salient kinetic Ϫ large uncertainty in the derived value for k9. feature of the ONOO formation is that, at a fixed pH, its rate Our suggestion of 1HNO deprotonation (reaction 7)asthe constant was independent of oxygen concentration throughout rate-determining step of ONOOϪ formation is further sup- the experimentally accessible range (Fig. 7, which is published as ported by the observation of the H͞D isotope effect on the supporting information on the PNAS web site). Thus, the reaction rate. The ONOOϪ formation rate constants were rate-determining step of this process is the reaction between the 2Ϫ Ϫ measured in H2O and D2O under conditions of Fig. 2 at 0.25 M N2O3 photolysis product and OH ; the reaction with O2 occurs alkali adjusted with NaOH and NaOD, respectively. At this in a step that is not rate limiting. Ϸ Ϫ alkalinity, reaction 7 dominates reaction 9 and kform k7[OH ]. Collectively, the results described are consistent with the ͞ ϭ Ϯ The ratio kform(H2O) kform(D2O) 3.0 0.1 was obtained, reaction sequence that begins with heterolytic photochemical 2Ϫ Ϫ which is consistent with proton involvement in the rate- cleavage of N2O3 , producing NO in its singlet state determining step. Ϫ 2Ϫ͑ϩ ͒ 3 1 Ϫ ϩ Ϫ The quantum yield of ONOO upon 266-nm laser pulse N2O3 hv NO NO2 . [5] 2Ϫ irradiation of N2O3 in O2-saturated 0.25 M NaOH was esti- Ϫ Ϸ As described above, 1NO is an extremely strong (pKa mated to be 0.5. The stoichiometry of peroxynitrite formation Ϸ given by reaction 4 shows that there are no channels for the 23) and, therefore, should be protonated by water nearly 2Ϫ N2O3 photochemical decomposition other than reaction 5. instantaneously 2Ϫ Thus, the quantum yield of the N2O3 photochemical cleavage 1 Ϫ ϩ 3 1 ϩ Ϫ is also close to 0.5. NO H2O HNO OH . [6] Ϫ Neutral solutions. In neutral solutions, the HN O monoanion Ϫ 2 3 In fact, the lifetime of 1NO is expected to be so short that not is the predominant trioxodinitrate species. The transient absorp- even its diffusion-controlled reactions with O2 should be able to tion spectra recorded after laser flash photolysis of O2-saturated Ϫ compete with reaction 6. Here we suggest that the rate- HN2O3 solution showed a characteristic peroxynitrite band determining step in the peroxynitrite formation is the deproto- around 300 nm (Fig. 3). This absorption decayed in seconds, Ϫ nation of 1HNO to 3NO , which is a much weaker base (pKa which is consistent with peroxynitrite lifetime at pH 7. To further Ϫ Ϸ11.4) than 1NO verify that the 300-nm band in Fig. 3 belongs to peroxynitrite, we Ϫ Ϫ resorted to a highly specific test based on the rapid decay of 1 ϩ 3 3 ϩ Ϫ HNO OH NO H2O. [7] peroxynitrite in bicarbonate solutions. At 20 mM HCO3 and pH Completing the process is the rapid, spin-allowed addition 7, the half- of the 300-nm band decreased to 20 ms, which reaction corresponds to the half-life of authentic peroxynitrite under these conditions (29). 3 Ϫ ϩ 3 3 Ϫ NO O2 ONOO . [8] The kinetics of peroxynitrite formation in nearly neutral solutions differed from those in alkali in two major ways. First, This mechanism accounts for the major reactivity features. the formation rate constant (kform) did not depend on pH in the Specifically, it predicts the independence of kform of [O2], as well 6–9 range and was orders of magnitude greater than at alkalin- as the linear dependence of kform on pH. The deviation from ities above pH 11. Second, kform linearly depended on the O2 linearity of the latter and the signal amplitude decrease in concentration (Fig. 7), suggesting that the rate-determining step solutions with pH Ͻ13 (Fig. 2) suggest the existence of an of peroxynitrite formation in neutral solutions involves a direct 1 Ϫ additional channel for HNO disappearance, which competes reaction between a nascent product of the HN2O3 photolysis with reaction 7. We could not satisfactorily account for these and O2. Apparently, a mechanism given by reactions 5–8 cannot effects by introducing a first-order channel for the 1HNO decay. account for the observations when the light-absorbing species is Ϫ Ϫ Moreover, a falloff in the ONOO yield with decreasing pH HN2O3 . analogous to that shown in Fig. 2 was not observed under To explain the data, we suggest two channels of the heterolytic

7342 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.112202099 Shafirovich and Lymar Downloaded by guest on September 23, 2021 Fig. 3. Transient absorption spectra measured at the indicated delay times Fig. 4. Transient absorption spectra recorded at the indicated delay times Ϫ Ϫ after laser pulse excitation of 0.6 mM HN2O3 at pH 7 (0.1 M phosphate). Open after flash photolysis of 0.3 mM HN2O3 in NO-saturated solution at pH 7 (0.1 symbols: O2-saturated solution; closed symbols: Ar-saturated solution. The M phosphate). (Inset) Kinetic trace recorded at 380 nm and showing formation ϭ ⌬ ϭ Ϫ solid line is calculated by using Eq. 13 with y 0.25 and C 0.052 mM; the of N3O3 ; note the time axis scale change. dashed lines are visual aids only. (Inset) Kinetic traces recorded at 315 nm for O2- and Ar-saturated solutions; the short negative-going signal at the time 3 Ϫ origin is caused by laser stray light. that NO was quantitatively scavenged by O2. The spectrum at the end of peroxynitrite accumulation can be described by the Ϫ equation photochemical cleavage of HN2O3 , one of which results in the ⌬ ϭ ͕ ␧ ͑ Ϫ͞ ͒ ϩ␧ ͑ Ϫ͒ formation of HNO in its triplet state, i.e., the reactions A␭ y ␭ ONOO ONOOH ␭ NO2 Ϫ͑ϩ ͒ 3 3 ϩ Ϫ Ϫ␧ ͑ Ϫ͖͒⌬ HN2O3 hv HNO NO2 [10] ␭ HN2O3 C, [13] Ϫ͑ϩ ͒ 3 1 ϩ Ϫ ⌬ Ϫ HN2O3 hv HNO NO2 . [11] where C is the concentration of HN2O3 decomposed by the Ϫ Ϫ laser flash and y ϭ ([ONOO ] ϩ [ONOOH])͞⌬C is the stoi- The protonation of N O2 reduces the NON from 2 3 Ϫ chiometric yield of peroxynitrite. The effective peroxynitrite Ϫ 2 to 1, because an H in HN2O3 resides on a nitrogen atom spectrum, ␧␭(ONOO ͞ONOOH), was constructed from the O Ϫ (7). This difference in the N N bonding may conceivably spectra of ONOO and ONOOH (pKa ϭ 6.6) according to their modulate the primary photochemical cleavage pathways. contribution at pH 7. The peroxynitrite yield y Ϸ0.25 was Branching into multiple pathways has been well documented for estimated by fitting this equation to the data in Fig. 3. Thus, the the photochemical decompositions of ; moreover, yield of 3NOϪ is also about 0.25 and the yield of 1HNO should 3 many of them very rapidly expel O as a result of the formally be close to 0.75. The estimated quantum yield of peroxynitrite spin-forbidden heterolytic bond cleavage (30–32). It is also (Ϸ0.2) is nearly the same as its stoichiometric yield. possible that reaction 10 is, in fact, spin-allowed. The triplet state Ϫ ؊ ؊ of NO2 is only 2.3 eV above the ground state (33) and the Formation of N3O3 by Reactions of HNO͞NO with NO. Neutral 266-nm photon energy (4.7 eV) is sufficient to produce 3HNO Ϫ solutions. In neutral solutions saturated with NO, the prompt and 3NO . As we argued previously, 3HNO is a strong acid and, Ϫ 2 bleaching of HN2O3 below 280 nm was followed by the growth therefore, should dissociate very rapidly in a buffered solution of an absorption band at 380 nm (Fig. 4), which subsequently Ϫ ϩ decayed with a 2.5-ms half-life (see Fig. 8, which is published as 3 3 3 ϩ [12] HNO NO H . supporting information on the PNAS web site). Both the ab- sorption spectrum and the lifetime were characteristic of the Peroxynitrite formation via reaction 8, which becomes the Ϫ N3O anion reported previously (17, 18, 34); reaction 3 was rate-limiting step, then follows. It should be noted that any 3 Ϫ CHEMISTRY 3 Ϫ suggested for the N O formation and decay in these reports. alternative chain of events that to NO within a few 3 Ϫ3 nanoseconds upon laser light absorption will explain the data. The kinetics of N3O3 formation consisted of two exponential components well separated in time (Fig. 4 Inset). The pseudo- The kinetics of absorbance increase at 315 nm in O2-saturated solution consisted of a fast rise, which could not be time resolved, first-order rate constants for both components (kfast and kslow) increased linearly with NO concentration. We have attributed and a slower growth with first-order rate constant kform (Fig. 3 Ϫ ␮ the fast process to the production of N3O3 by the rapid sequen- Inset). The transient spectrum at the end of the fast rise (0.05 s 3 Ϫ in Fig. 3) could be assigned to the superposition of absorption by tial additions of two NO radicals to the NO anion generated 3 Ϫ Ϫ in reactions 10 and 12, i.e., the reactions NO and bleaching of HN2O3 apparent below 280 nm. Indeed, very similar spectra appeared on this time scale upon flash 3 Ϫ ϩ 3 Ϫ Ϫ NO NO N2O2 [14] photolysis of Ar-saturated HN2O3 solutions (Fig. 3), where no peroxynitrite was formed. The slower absorption growth at 315 Ϫ ϩ 3 Ϫ N2O2 NO N3O3 . [15] nm corresponded to reaction 8; from the linear dependence of ϭ Ϯ ϫ 9 Ϫ1⅐ Ϫ1 9 kform on [O2] the rate constant k8 (2.7 0.2) 10 M s The effective bimolecular rate constant of (2.3 Ϯ 0.2) ϫ 10 was obtained (see Fig. 7). MϪ1⅐sϪ1, which approximately corresponds to the slower of these The transient absorbance amplitude at 315 nm upon the reactions, was determined from the slope of the kfast vs. [NO] plot completion of peroxynitrite formation was independent of ox- (see Fig. 9, which is published as supporting information on the ygen concentration in the 0.3 to 1.3 mM range, which indicates PNAS web site). The relative contributions of the fast and slow

Shafirovich and Lymar PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7343 Downloaded by guest on September 23, 2021 Scheme 1.

Ϫ constants reported here are expected to be small. The intercon- Fig. 5. Dependence of the N3O3 formation rate constant (kapp)onthe version between 3NOϪ and 1HNO (reaction 7) is of special solution pH (adjusted with NaOH at pH Ն 12 and 0.1 M borate at pH 10). (Inset) Ϫ interest because it gives a unique example of a spin-forbidden pH independence of the slow step rate constant (kslow) for N3O3 formation in ϭ ϫ 4 Ϫ1 (nonadiabatic) proton transfer between a pair of small mole- buffered solutions. Both solid lines correspond to Eq. 17 with a 1.1 10 s Ϫ Ϫ Ϫ ϭ 1 and b ϭ 5.3 ϫ 104 M 1⅐s 1. All data are for NO-saturated solutions. cules. The equilibrium constant K7 400 M can be calculated from pKa(1HNO͞3NOϪ) ϭ 11.4. Using the experimental value ϭ ϫ 4 Ϫ1⅐ Ϫ1 Ϫ for k7 4.9 10 M s , we estimate the rate constant for the 3 Ϫ pathways to the N3O3 formation measured from the amplitudes NO decay through the spin-forbidden protonation by water of these components (Fig. 4 Inset) were about 25% and 75%, Ϫ ϩ 3 1 ϩ Ϫ ؊ 3 respectively. These values are in agreement with the yields of NO H2O HNO OH [ 7] 3 Ϫ 1 NO and HNO estimated above for the reaction sequence ϭ ϫ 2 Ϫ1 3 Ϫ to be kϪ7 1.2 10 s , i.e., the NO lifetime in water is 10–12 from the data on peroxynitrite formation. We have, Ϫ several milliseconds. This long lifetime allows the 3NO anion to therefore, concluded that the rate-determining reaction in the Ϫ participate in reactions with O and NO. Without the spin slow process of the N O formation is the addition of NO to 2 3 3 prohibition, anions with similar basicity equilibrate with water 1HNO much more rapidly. Recently, the spin-forbidden protonation of Ϫ 1 ϩ 3 Ϫ ϩ ϩ 3NO has been directly observed in the gas phase, albeit for HNO NO N2O H , [16] 2 much stronger than water (35). The independent estimates Ϫ 15 ϭ for the upper limit of pKa(1HNO͞3NO ) Ͻ 14.3 and for the which is followed by rapid reaction . The rate constant k16 Ϫ Ϯ ϫ 6 Ϫ1⅐ Ϫ1 0 ͞3 ϾϪ (5.8 0.4) 10 M s was obtained from the kslow vs [NO] lower limit of E (NO NO ) 1 V, which are in agreement dependence (see Fig. 9). At a constant concentration of NO, with the values in Fig. 1, can be made by using our kinetic data kslow was independent of pH in the 4 to 10 range (Fig. 5 Inset). (see Note 2, which is published as supporting information on the Alkaline solutions. In alkaline solutions, the fast process of PNAS web site). Ϫ Ϫ Another potentially important spin-forbidden reaction could N3O3 formation was not observed and only the slow N3O3 accumulation occurred as a single exponential step. Both the to peroxynitrite Ϫ yield of N O and the rate of its subsequent decay were 3 3 1HNO ϩ 3O 3 ONOOH. [18] pH-independent above pH 10 and remained the same as at pH 2 7. In contrast, the apparent pseudo-first-order rate constant for Ϫ Although this reaction should be exoergonic by 100 kJ͞mol, we N3O3 formation (kapp) steeply increased with alkalinity (Fig. 5) found no evidence for its occurrence, which allowed an estimate in a manner similar to that shown in Fig. 2 for ONOOϪ of the upper limit for k18. From the relative amplitudes in Fig. 2 formation. The pH dependence of k conformed to the ϽϽ 1 ϽϽ ϫ app it follows that k18[O2] k9[ HNO]0 and, therefore, k18 3 expression 105 MϪ1 ⅐sϪ1. A more plausible mode of oxygen reactivity is ϭ ϩ ͓ Ϫ͔ atom transfer kapp a b OH [17] 1HNO ϩ 3O 3 NO ϩ HO , [19] with a ϭ (1.1 Ϯ 0.1) ϫ 104 sϪ1 and b ϭ (5.3 Ϯ 0.5) ϫ 104 MϪ1⅐sϪ1. 2 2 The second term describes the effect of alkalinity and can be ͞ Ϫ which is not only energetically favorable by 25 kJ mol, but is also attributed to the generation of 3NO by deprotonation of 1HNO spin-allowed. Both reactions 9 and 19 have been studied in the ͞ Ϸ ϫ 5 (reaction 7). Indeed, within the experimental accuracy, the b gas phase and the ratio (k9 k19)gas 2 10 at 298 K can be value is identical to the k7 value determined above from the calculated from the published data (36). If the ratio were the peroxynitrite formation experiments. The formation of 3NOϪ is Ϫ same in water, reaction 9 would always dominate under our followed by very rapid reactions 14 and 15, leading to N3O3 . experimental conditions, making reaction 19 undetectable. For ϭ Likewise, the term a corresponds to reaction 16, i.e., k16[NO] more on water vs. gas rate comparison see Note 3, which is a in the NO-saturated solutions. Under these conditions, side published as supporting information on the PNAS web site. reaction 9 is too slow to play a role. Thus, the flash photolysis The mechanistic interpretation of the experimental data data for both O2- and NO-containing solutions could be ac- based on Fig. 1 and shown in Scheme 1 provides a novel view on counted for on the basis of the common reactions 5–7 and 10–12. the interplay between the spin states and reactivities in the HNO͞NOϪ system. At the same time, it challenges the inter- Mechanistic Considerations. The mechanistic information ob- pretation of the early pulse radiolysis work (17, 18). The tained in this work is summarized in Scheme 1, where reactions disagreements pertain to pKa of HNO, to the existence of rapid Ϫ ª ϩ Ϫ Ϫ are numbered as in the text. Their common feature is that at least equilibrium N2O2 NO NO , and to the rate of N3O3 one of the reaction partners is an uncharged species; conse- formation from NOϪ in NO-saturated solutions. It appears that quently, the effects of medium ionic strength on the rate reexamination of the related experiments in the literature is

7344 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.112202099 Shafirovich and Lymar Downloaded by guest on September 23, 2021 required to resolve these matters. Similarly, the pKa(1HNO͞ Can peroxynitrite be produced through intermediacy of 3NOϪ 3NOϪ) value of 7.2 derived from the quantum mechanical and 1HNO in oxygenated tissues? Although our results do not calculations (22) is incompatible with our data, suggesting that rule out this possibility, they impose limitations on the reaction further computational refinements may be necessary. Finally, a conditions that may lead to peroxynitrite. We have shown that 3 Ϫ value that is almost 1,000 times greater than determined in addition of O2 to NO is essentially diffusion controlled and 3 Ϫ present work was suggested for k9 (28). However, this value is gives peroxynitrite. However, generation of NO requires a based largely on the early pulse radiolysis results that we are now very strong reductant, which is unlikely to survive other reactions questioning. in biological milieus, unless embedded in a hydrophobic envi- ronment with limited access to dissolved species. Such environ- ϩ Biological Implications. If nitrogen( 1) is indeed produced in ments may include hydrophobic pockets in proteins and biolog- 1 biological systems, it must be present in the form of HNO at ical membranes. It is much more probable that biological systems 1 physiological pH. As a small neutral , HNO should be can generate 1HNO rather than 3NOϪ because it requires only able to freely traverse biological membranes, a property that can 1 a moderate reductant. Direct addition of O2 to HNO that could 1 make a physiological role of HNO very significant. We estimate yield peroxynitrite is spin-forbidden and, as we have shown, 0 ϩ͞1 ϷϪ the reduction potentials E (NO, H HNO) 0.14 V and cannot be rapid, if it occurs at all. It appears that the possibility 0 1 ϩ͞ Ϸ E ( HNO, 2H NH2OH) 0.7 V; the corresponding values for of peroxynitrite formation depends greatly on the magnitude of pH 7 are Ϫ0.55 V and 0.3 V, all vs. NHE. With these potentials, 1 the rate constant for this reaction, which remains unknown. In HNO can engage in redox reactions with a number of diverse this regard, coordination to paramagnetic metal (including biological reductants and oxidants. However, in NO-rich envi- Ϫ those in proteins) could conceivably facilitate formally spin- ronments, consecutive addition of NO to form N3O3 (reactions forbidden peroxynitrite formation from 1HNO and O . 16 and 15) can be envisioned as the major sink for 1HNO. In this 2 respect the spontaneous decomposition of Angeli’s salt provides 1 We thank Drs. Merenyi and Poskrebyshev for insightful discussions. a convenient method to generate HNO in the absence of NO. Work at New York University was supported by a grant from the Kresge At the same time, our results underscore the importance of using Foundation. Research at Brookhaven National Laboratory was carried freshly prepared alkaline stocks of Angeli’s salt to avoid con- out under the auspices of the U.S. Department of Energy under Contract tamination with peroxynitrite through reactions 7 and 8 upon DE-AC02-98CH10886 from the Division of Chemical Sciences, Office of aging of the stock. Basic Energy Sciences.

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